Discussion

- Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior

Significant progress has been made in unraveling the molecular mechanism underlying the mammalian circadian system. The core molecular circuitry of opposing interlocking transcriptional feedback loops has been defined as the fundamental basis of the circadian clock [2,74]; however, with subsequent discovery of additional molecular components of the circuitry [75–78], the complexity and network intricacy of the clock system are becoming apparent [2,11,50,79]. Ultimately, we want to understand how these cell-autonomous circadian oscillators interact in multicellular organisms to regulate physiology and behavior [80]. Thus, to elucidate the mechanisms governing the hierarchical nature of mammalian circadian timing, it is necessary to develop genetic tools to manipulate circadian genes in a conditional and tissue-specific manner in vivo.

We adapted an in vivo transgenic method, the tTA system, to regulate Clock gene expression in a conditional and reversible manner. This is a significant technical milestone that has not been previously demonstrated in the mammalian circadian system. In this report, we generated an SCN- and brain-enriched tTA-expressing transgenic line, which can transactivate any transcript of choice when crossed to tetO-responsive transgenic lines. We also produced target lines that can express a ClockΔ19 mutant allele or Clockwt allele in a tissue-specific manner when crossed to a transactivator transgenic line. The ClockΔ19 allele is a dominant-negative mutation (antimorph) [56] and is an ideal allele to validate the tTA system for circadian experiments for several reasons. First, the Clock gene, with its heterodimeric partner Bmal1/Mop3, is one of the primary transcriptional activators of the circadian transcriptional autoregulatory feedback loop. Second, the ClockΔ19 mutation in mice perturbs the circadian system and causes a significantly altered running-wheel rhythm that is clearly distinguishable from WT [56,57,65,66]. The antimorphic nature of this mutation, which has a phenotypic effect that is antagonistic with the normal allele, allows manifestation of the mutant phenotype in the presence of the WT allele. Third, we have previously demonstrated not only that transgenic expression of WT Clock can completely rescue circadian period and amplitude in Clock mutant mice but also that overexpression of the Clockwt TG ubiquitously results in period shortening beyond the normal WT values [66]. Fourth, Clock gene expression in the SCN is constitutive, which is easier to mimic with tTA system. Finally, all circadian clock mutants that have been analyzed in mammals thus far have been germline mutations (either ENU-induced or gene targeted), and therefore the developmental consequences of these mutations have not been addressed. For all of these reasons, we set out to test conditional expression of Clock on circadian behavior.

Interestingly, the circadian periodicity of Scg2::tTA/tetO::ClockΔ19-HA double transgenic mice provides a real-time readout of the transactivation state of this system. This allowed us to follow the kinetics of Dox regulation on a day-to-day basis and revealed that high doses of Dox (2 mg/ml) required many months of time to washout and reverse. This then gave us the opportunity to find optimal Dox dose treatments for both inactivation and reversal of tet-dependent transactivation in the brain of mice. SCN-directed expression of the dominant-negative ClockΔ19 TG lengthens the circadian free-running period, whereas expression of the Clockwt TG shortens the circadian rhythm. Furthermore, we showed that temporal and spatial control of TG expression can revert the phenotype from a mutant to a WT state within individuals, and vice versa, with a low concentration of Dox treatment, so that experiments can be performed in a longitudinal fashion. This is akin to transplant experiments [17–19] with the added ability to reverse the procedure. Moreover, unlike transplant experiments, which only allow for the receiving of intact humoral signals, our system also retains intact synaptic connections of the SCN. Finally, the expression of the TG affects not only the free-running activity rhythm but also other expected circadian behavioral responses, such as phase shifts to discrete light pulses. Thus, the transgenic mice described in this report are a valuable tool and will facilitate investigation of the functional relationship between central and peripheral clocks.

The validation of the tTA system for conditional TG expression in a variety of cell types and tissues has made it the tool of choice for mammalian system research. Arguably, some of the most significant contributions were made by several groups utilizing the Tet-system in vivo to study the effects of conditional TG activation and repression on various neurobiological process [35–39,43,45,81,82]. However, our study differs uniquely from previous Tet system applications in several ways. First, this is the first report on the mammalian circadian system where an SCN/brain-driver is used to conditionally regulate clock genes in vivo. Only a handful of studies reported have used the Tet system in the brain and, thus, the availability of brain-specific drivers is very limited. Furthermore, no drivers have previously been shown to function in the SCN. Second, our study demonstrates that circadian locomotor activity records give us a unique opportunity to have a daily readout of the transactivation state in a noninvasive manner. We suspect that this is likely due to a combination of the shorter half-life of the target protein, CLOCK, and our optimization of the Dox dosage used. Thus, we show that CLOCK is an excellent indicator for the kinetics of Dox-dependent induction/suppression in the brain. Third, we show that the standard dose often used in Tet regulation studies (e.g., 2 mg/ml) is excessively high. This leads to very slow kinetics of washout and slow (weeks to months) reactivation of the TG after standard doses of Dox treatment [49]. To date, only one study has examined a time course of TG expression in the brain tissues using luciferase activity as an indicator of the TG expression and has suggested administering a 100-fold lower dose (50 μg/ml) [36]. In our study, we demonstrate that even a 200-fold lower dose of 10 μg/ml in drinking water is sufficient to cross the blood-brain barrier and is equally effective in turning off the TG, and subsequently regulate behavioral state. With a lower dose, our results reveal that the washout is rapid and, thus, multiple induction and suppression cycles of the TG can be achieved within subjects with minimal time loss and cost. Furthermore, the effectiveness of the low Dox dosage is not driver specific. We have found that another SCN- and brain-enriched driver to be inducible and reversible using the same low dose administration (unpublished data). Finally, this study provides an important set of transgenic mouse resources for the circadian research community.

Exploitation of these transgenic lines along with existing genetic allelic series of circadian genes may yield fundamental insights into the mechanism by which circadian pacemaker systems transmit information to control physiology and behavior. In addition, by using peripheral tissue-specific drivers, manipulations using the tTA system can yield a wealth of knowledge on physiological processes tied to the circadian machinery such as cell division, heme biosynthesis, tumor suppression, metabolism, and bone remodeling [83–87]. For example, we recently reported a dissection of tissue-specific functions of the mammalian clock protein BMAL1 using the SCN- and brain-enriched driver line, which we describe here, and a muscle-specific driver line. We showed that distinct tissue-specific phenotype in Bmal1-null mice can be rescued using the tTA system [88]. Moreover, tetracycline-dependent genetic tools can also assist in elucidating unexpected subtle phenotypes found in knockout mice of some essential clock genes, such as Rev-erbα and Clock [89,90], and address our current criteria for definition of primary clock components [3]. Besides being potentially useful for such studies, the tTA system may be a great resource for the discovery and in vivo validation of novel candidate genes that may be involved in the central SCN oscillator and the output pathway. The successful manipulation of conditional TG expression in the SCN and brain in these studies will lay the groundwork for the development and adaptation of other tools such as the Cre/Lox system for tissue-specific knockout and conditional inactivation of circadian genes. Furthermore, by developing additional SCN subregional-specific drivers, we can begin to decipher the function of the cellular heterogeneity of the mammalian SCN and to understand how these pacemaking neurons are organized to mediate synchrony within the SCN and the whole animal. The flexibility of the tTA system provides a means to dissect the cellular and behavioral networks in the mammalian circadian system.